The attachment of cells and proteins to substrates is a well-known problem that has presented itself in a number of contexts. For example, in cell cultures to produce antibodies, fibroblasts attach to extracellular matrix proteins bound to the tissue culture substrate. Similarly, in urinary catheters, bacterial cells attach to the walls of the catheter; in arterial catheters, platelets attach to the tip of the catheter; and in contact lenses, proteins coat the surfaces of the lenses.
Various bioadhesives are known in the art. U.S. Pat. No. 4,615,697, issued to Robinson et al., defines a bioadhesive as a material that requires a force of at least about 50 dynes/cm2 to separate two adhered, freshly excised pieces of rabbit stomach, following the procedure disclosed therein. The bioadhesive disclosed in Robinson et al. is a water-swellable, but water insoluble, fibrous, cross-linked carboxy-functional polymer.
Various attempts to ameliorate the problem of attachment of cells and proteins to substrates have been employed, but none have been found to be satisfactory. It would be desirable to solve this problem using a biocompatible substance that is adherent to substrates and inhibits cellular and protein attachment.
Certain cells, such as macrophages and fibroblasts, are referred to as xe2x80x9csubstrate-dependent cellsxe2x80x9d because they are active and proliferate only when attached to a surface or substrate. The attachment occurs via a family of proteins (xe2x80x9cattachment molecules or proteinsxe2x80x9d), such as vitronectin and fibrinectin, which are found in the extracellular matrix. A surface that is coated with a material that is strongly adhesive may inhibit the attachment of substrate dependent cells by blocking attachment of extracellular matrix proteins. Hence, adhesive materials, as described herein, are useful in compositions or can form devices that inhibit the attachment of certain proteins and certain types of cells.
The present invention features a method of inhibiting cellular attachment to substrates. The invention is based, in part, on the discovery of adherent coatings of N,O-carboxymethylchitosan (xe2x80x9cNOCCxe2x80x9d), and in particular that adherent coatings of NOCC may be applied to various substrates, such as mammalian tissue, so as to inhibit attachment of other cells, such as substrate dependent cells. Further, it has been discovered that these adherent coatings of NOCC may be used in other areas where inhibition of cell or protein attachment is desirable, such as in the preparation of cell populations, on medical devices, and with cell-based products. The invention also has application to the inhibition of the attachment of proteins to surfaces.
The present invention provides a composition that is adherent to a variety of synthetic materials and mammalian tissues. The present invention also provides a method of inhibiting cellular and protein attachment to a substrate by applying adherent coatings of NOCC to the substrate such that the attachment of cells and proteins is inhibited. The amount of adherent NOCC in the composition should be effective to inhibit the attachment of substrate-dependent cells, preferably in a concentration of 0.05-5%(w/v), most preferably in a concentration of 0.1-2.5%(w/v).
In one embodiment, the invention provides a composition and method of inhibiting attachment of substrate-dependent cells to a substrate by applying a composition containing adherent NOCC to a substrate such that attachment of substrate-dependent cells is inhibited. The method may be applied to inhibit substrate-dependent cell attachment to mammalian tissue, medical devices, fermentation units, bioreactors and solid supports. In preferred embodiments, the substrate-dependent cells which are inhibited include fibroblasts, macrophages, epithelial cells, and endothelial cells.
In another embodiment, the invention provides a composition and method of inhibiting attachment of proteins to a substrate by applying a composition containing adherent NOCC to a substrate such that attachment of proteinaceous material is inhibited. The method may be applied to inhibit protein attachment to contact lenses, medical devices, fermentation units, bioreactors and solid supports.
In another embodiment, the invention may be used in a method of obtaining a population of cells, e.g. mammalian cells, by supplementing culture media with adherent NOCC, growing the population of cells in the supplemented media, and allowing the cells to grow or differentiate, such that substrate-dependent cells do not proliferate within the cell population.
In another embodiment, the invention provides a method of obtaining cells suitable for use in protein or antibody production by supplementing culture media with adherent NOCC and growing the cells in the supplemented media, such that intercellular attachment (or clumping) within the cell population is inhibited and production of proteins or antibodies is enhanced.
In yet another embodiment, the invention provides a method of inhibiting attachment of inflammatory cells and platelets to a medical device by coating said device with a composition containing adherent NOCC, such that platelet or inflammatory cell attachment to the medical device is inhibited. In preferred embodiments, the internal medical device is either a stent or shunt. In other preferred embodiments, the inflammatory cell includes fibroblasts, macrophages, and monocytes.
In still another embodiment, the invention includes a method of inhibiting fibroblast attachment in a cell-based product in contact with a solid support by introducing adherent NOCC into the cell based product such that fibroblast attachment is inhibited.
In another embodiment, the invention provides a composition and method of delivering drugs, proteins, and other therapeutic agents from an adhesive device or composition that is adherent to soft (mucosal or non-mucosal) tissue or hard tissue. In preferred embodiments, the adherent delivery device can be used as a buccal, oral, vaginal, inhalant, or the like delivery system. The device can be in a variety of forms including solutions, creams, pellets, particles, beads, gels, and pastes.
The present invention relates to the inhibition of cellular and protein attachment to various substrates. The method of the invention uses an adherent coating of N,O-carboxymethylchitosan (xe2x80x9cNOCCxe2x80x9d) which provides unexpected benefits in inhibiting cellular and protein attachment.
NOCC is a derivative of chitin, which is found in the shells of crustaceans and many insects. Chitin and its derivatives are normally biocompatible, naturally resorbed by the body, and have previously been used for sustained drug release, bone induction and hemostasis (Chandy and Sharma, Biomat. Art. Cells and Immob. Biotech. 19:745-760 (1991); Klokkevold, P. et al., J. Oral Maxillofac. Sur. 50:41-45 (1992)). Due to its prevalence, chitin may be obtained relatively cheaply, largely from waste products. As disclosed in U.S. Pat. No. 4,619,995, issued to Hayes, NOCC has carboxymethyl substituents on some of both the amino and primary hydroxyl sites of the glucosamine units of the chitosan structure. NOCC may be used in an uncrosslinked form as a solution or may be cross-linked or complexed into a stable gel. Because of its advantageous physical properties, and its relative low cost, NOCC presents advantageous properties for use in inhibiting cellular and protein attachment.
Definitions
The term xe2x80x9cinhibit,xe2x80x9d or any form thereof, is defined in its broadest sense and includes minimize, prevent, repress, suppress, curb, constrain, restrict and the like.
The terms xe2x80x9cadherent NOCCxe2x80x9d or xe2x80x9can adherent coating of NOCCxe2x80x9d mean a coating or composition of NOCC that exhibits an adhesion between freshly excised tissue of at least about 100 dynes/cm2, using the procedure described in Example 1.
The term xe2x80x9csubstratexe2x80x9d refers to any object to which cells can attach. Examples of substrates include, without limitation, mammalian tissue (including both hard tissue, such as bone, and soft tissue, such as mucosal and non-mucosal tissue), non-mammalian tissue, mammalian and non-mammalian cells (including both eukaryotic and prokaryotic organisms), medical devices, fermentation units, bioreactors, and solid supports, such as cell culture plates.
The term xe2x80x9csubstrate-dependent cellsxe2x80x9d means cells that are only active when attached to a substrate. Examples of substrate dependent cells include, without limitation, fibroblasts, macrophages, epithelial cells, somatic cells, and endothelial cells.
The term xe2x80x9cmedical devicexe2x80x9d means any device which is implanted in the body for medical reasons or which has a portion of the device extending into the body (like a catheter) as well as devices which provide a medical benefit when attached to, or are in contact with, the body. Examples of medical devices include, without limitation, catheters, contact lenses, stents, shunts, breast implants and pacemakers.
The term xe2x80x9cinflammatory cellxe2x80x9d means a cell involved in the non-specific immune response to any type of body injury. Examples of inflammatory cells include, without limitation, fibroblasts, macrophages, eosinophils, neutrophils, monocytes and lymphocytes.
The term xe2x80x9ccell-based productxe2x80x9d means any product that contains cell. Examples of cell-based products include, without limitation, blood, plasma, aliquots of cell cultures, and the like.
The invention provides a method of inhibiting attachment of substrate-dependent cells or proteins to a substrate by applying a composition containing adherent NOCC to a substrate such that attachment of the substrate-dependent cells or protein is inhibited. In preferred embodiments, the method is applied to inhibit substrate-dependent cell attachment to mammalian tissue, medical devices, fermentation units, bioreactors and solid supports. In preferred embodiments, the substrate-dependent cells which are inhibited include fibroblasts, macrophages, epithelial cells, and endothelial cells.
The invention also may be used in a method of obtaining a population of cells, e.g. mammalian cells, by supplementing culture media with adherent NOCC, growing the population of cells in the supplemented media, and allowing the cells to grow or differentiate, such that substrate-dependent cells do not proliferate within the cell population.
The invention further provides a method of increasing the efficiency of protein or antibody production by supplementing culture media with adherent NOCC and growing the cells in the supplemented media, such that intercellular attachment within the cell population is inhibited and production of protein or antibodies is enhanced.
The invention also provides a method of inhibiting attachment of inflammatory cells or proteins to a medical device by coating said device with a composition containing adherent NOCC, such that inflammatory cell or protein attachment to the medical device is inhibited. In preferred embodiments, the medical device is a catheter, a contact lens, a stent, pacemaker, breast implant, or a shunt. The method is useful for preventing attachment of a variety of inflammatory cells including fibroblasts, macrophages, monocytes, as well as proteins such as albumin.
In still another embodiment, the invention includes a method of inhibiting fibroblast attachment in a cell-based product in contact with a solid support by introducing the cell based product to an adherent coated solid support such that fibroblast attachment is inhibited.
The adherent NOCC used in the present invention may take many forms. For example, adherent NOCC may be used in a solution, a hydrogel, a paste, a rehydratable film, cream, foam, or a sponge. These forms are prepared by methods well known to those of ordinary skill in the art.
The adherent NOCC used in the present invention may be the parent compound or may be cross-linked. Cross-linked adherent NOCC may be either covalently cross-linked or ionically cross-linked. Various methods of cross-linking NOCC are known in the art and are within the scope of this invention. In addition, the degree to which the adherent NOCC is cross-linked may be optimized for specific applications by one of ordinary skill without undue experimentation. It has been found that the degree of cross-linking is roughly inversely proportional to the adhesiveness of the coating. That is, the greater the degree of cross-linking of the adherent NOCC, the lesser degree of adherence. In preferred embodiments, the degree of cross-linking is less than 1:5 (moles cross-linking agent to moles, NOCC monomer), more preferably between 1:100 and 1:1000 on a molar basis.
The bioadhesive strength of several adherent NOCCs was compared to that of polycarbophil, a cross-linked acrylic acid polymer available from B. F. Goodrich. As more fully described in Example 1, solutions of low and high viscosity NOCC were prepared, as well as hydrogels of high viscosity NOCC. The bioadhesive was applied to stomach and cecal tissue samples and the bioadhesive strength was measured according to a modified version of the procedure disclosed in U.S. Pat. No. 4,615,697, which is hereby incorporated by reference. The transfer of polymer to both tissue surfaces indicated that the adhesive force of the polymer exceeded the cohesive force. A summary of results appears in Tables 1 and 2. In preferred embodiments, the bioadhesive strength of adhesive NOCC coatings of the invention is desirably greater than at least about 1000 dynes/cm2, more preferably greater than at least about 2000 dynes/cm2, and most preferably greater than at least about 3000 dynes/cm2.
Both the low viscosity and high viscosity NOCC polymer solutions in citrate buffer behaved similarly to polycarbophil when applied as a coating to the mucosal surface of stomach tissue (Table 1). This was also true for similar solutions of NOCC using phosphate buffered saline instead of citrate buffer as well as non-mucosal, cecal tissue (Table 2). It was observed that as NOCC was cross-linked the cohesion of the materials increased and the adhesion decreased. The loss of adhesion was dependent on the extent of cross-linking. These findings are likely attributable to the fact that cross-linking adherent NOCC introduced more structure into the polymer, which consequently restricted interactions with the tissue surface. The cross-linking also joined the polymer chains together, resulting in increased cohesiveness.
The ability of NOCC to adhere to bone tissue was also studied. The results indicate that NOCC adheres to bone tissue. After the third wash, 9.5+10xe2x88x923xc2x10.002 xcexcL/mm2 (or about 0.1 xcexcg NOCC/mm2) of 125I labeled NOCC remained adhered to the rat femur.
Surprisingly, the adhesive NOCC coatings of the present invention have been shown to inhibit cellular attachment of substrate dependent cells. The adherent NOCC coatings of the present invention thus have applicability in a multitude of areas. In addition, adherent NOCC coatings may be applied to either hard or soft mammalian tissue, such as bone or stomach tissue. Alternatively, adherent NOCC coatings may be applied to non-biological substrates, such as medical devices and solid supports. Examples of such substrates include stents, shunts, contact lenses, microtiter plates, and cell culture plates.
Typically, fibroblasts in a cell or tissue culture adhere to extracellular matrix (ECM) proteins that are bound to the culture substrate (usually plastic). The ECM proteins in culture typically come from the culture medium, which is supplemented with serum to provide these proteins as well as other factors necessary for cell growth. Alternatively, if there are no ECM proteins in the culture medium, fibroblasts will secrete their own ECM proteins and adhere to them. A normal, adhered fibroblast has a very characteristic morphology: it flattens and exhibits cellular appendages or processes extending from the cell over the substrate surface, which indicates fibroblast adherence to the substrate.
The present invention takes advantage of the observation that substrate-dependent cells, e.g. fibroblasts, plated in tissue culture media in the presence of adherent NOCC coating do not have the characteristic morphology and do not exhibit processes indicating attachment of the cell.
Initial observations of fibroblast morphology in the presence or absence of adherent NOCC in the medium, revealed that when fibroblasts were plated in serum-free media without adherent NOCC they consistently displayed an xe2x80x9cadherentxe2x80x9d morphology, viz. the cells were flattened with complex processes. As described more fully in Example 3, fibroblasts were plated on tissue culture plates, either in the presence or absence of adherent NOCC, under four different coating conditions. Irrespective of the coating treatment, approximately 80% of cells observed looked like normal cultured fibroblasts in the absence of adherent NOCC. In contrast, when fibroblasts were plated in the presence of adherent NOCC, the number of cells displaying the adherent morphology was significantly reduced. In fact, in the instance where no ECM proteins were present, no cells adhered in the presence of adherent NOCC. Where ECM proteins were present, some cell adherence was observed, but the adherence was significantly less than that which occurred in the absence of adherent NOCC.
Hyaluronic acid (HA) was also tested in this system, to determine whether it had similar effects to NOCC. When a similar morphological examination was performed on cells plated in sfRPMI (serum free or protein free RPMI medium) containing 0.1% HA, it was observed that the HA did not have the same effect on fibroblast morphology.
Fibroblast adherence was also measured quantitatively, using a 51Cr adhesion assay (the 51Cr release assay). The results confirmed that adherent NOCC blocks adhesion of 3T3 fibroblasts to plastic, by more than 90% using this assay. This result, taken together with the previous work, suggests that adherent NOCC adheres to the substrate and interferes with the deposition of ECM proteins in a competitive manner.
A competitive assay was performed using media supplemented with varying concentrations of fetal calf serum (FCS), which contains the ECM proteins of interest, and in the presence or absence of NOCC. As expected, it was found that the presence of FCS in the plating medium reversed the inhibitory effect of adherent NOCC on fibroblast adhesion in a dose dependent manner, where 10% FCS fully restored binding of 3T3 to the plates in the presence of NOCC. There are two possible explanations for this effect: 1) adherent NOCC prevents fibroblast adhesion to the ECM proteins which bind to the plate, or 2) adherent NOCC prevents the binding of ECM proteins to the plate in a competitive manner.
To address these possibilities, tissue culture plates were pre-coated with serum-free medium containing varying concentrations of FCS. The presence of adherent NOCC did not interfere with the adhesion of fibroblasts to the FCS coated plates, which confirmed that adherent NOCC does not inhibit adhesion of fibroblasts to ECM proteins already deposited on the plate. This result was confirmed by coating tissue culture plates with an adherent NOCC coating. The results demonstrate that fibroblast adherence to adherent NOCC coated plastic is eliminated in the presence of FCS. This result supports the hypothesis that adherent NOCC binds to the plastic plate surface and prevents the deposition or attachment of ECM proteins. Thus, adherent NOCC may inhibit cellular attachment by preventing the deposition of ECM proteins rather than by inhibiting the adhesion of fibroblasts to the ECM proteins. In the absence of the ECM protein network, fibroblasts are unable to bind to a substrate.